The goal of the proposed research is to bioengineer a temporary oxygen carrier for use as an alternative to banked blood. There is currently a need for blood substitutes because of shortages in the blood supply and potential infection spread through donated blood, especially in countries lacking a clean blood supply. Temporary blood substitutes currently under investigation, based on hemoglobins and perfluorocarbons, have yet to meet with significant regulatory success due to side effects. This project will focus on using members of the Heme Nitric oxide/OXygen (H-NOX) protein family as scaffolds for the development of novel temporary oxygen carriers. Temporary oxygen carriers must exhibit long-term stability under standard storage conditions, stability in plasma, and high selectivity for oxygen over nitric oxide, as nitric oxide scavenging leads to hypertensive side effects. H-NOX proteins display ligand selectivity between oxygen and nitric oxide, making them prime candidates for engineering. Amino acid mutations will be generated in the heme distal pocket to increase the ligand selectivity of these proteins, as well as slow the autooxidation rate of the heme. Directed evolution will also be performed on H-NOX proteins to generate libraries of random mutants with altered ligand binding properties. A selection assay and screen will be developed to identify mutants with the desired oxygen binding characteristics. The evolved H-NOXs will then be subjected to the selection assay and screen and those mutants with improved properties will be fully spectroscopically characterized using resonance Raman and UV-visible spectroscopy, as well as stop-flow and laser flash photolysis to measure ligand binding kinetics. The most promising mutants will be tested for plasma compatibility and chemically modified to increase their stability in plasma. The design of novel blood substitutes is very important due to blood shortages and risk of disease transmission from donated blood. The goal of this research is to develop a new blood substitute, based on a known protein scaffold, that has diminished side effects and improved efficacy as compared to current products.